ABSTRACT
Shank3, a key molecule related to the development and deterioration of autism, has recently been found to downregulate in the murine brain after ischemia/reperfusion (I/R). Despite this discovery, however, its effects on neuronal injury and the mechanism underlying the effects remain to be clarified. To address this, in this study, based on genetically modified mice models, we revealed that the expression of Shank3 showed a time-dependent change in murine hippocampal neurons after I/R, and that conditional knockout (cko) of Shank3 in neurons resulted in aggravated neuronal injuries. The protective effects of Shank3 against oxidative stress and inflammation after I/R were achieved through direct binding STIM1 and subsequent proteasome-mediated degradation of STIM1. The STIM1 downregulation induced the phosphorylation of downstream Nrf2 Ser40, which subsequently translocated to the nucleus, and further increased the expression of antioxidant genes such as NQO1 and HO-1 in HT22 cells. In vivo, the study has further confirmed that double knockout of Shank3 and Stim1 alleviated oxidative stress and inflammation after I/R in Shank3cko mice. In conclusion, the present study has demonstrated that Shank3 interacts with STIM1 and inhibits post-I/R neuronal oxidative stress and inflammatory response via the Nrf2 pathway. This interaction can potentially contribute to the development of a promising method for I/R treatment.
Subject(s)
Brain Ischemia , Reperfusion Injury , Mice , Animals , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Reperfusion Injury/genetics , Reperfusion Injury/metabolism , Oxidative Stress , Brain Ischemia/genetics , Brain Ischemia/metabolism , Inflammation/genetics , Inflammation/metabolism , Reperfusion , Neurons/metabolism , Apoptosis , Microfilament Proteins/metabolism , Microfilament Proteins/pharmacology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolismABSTRACT
Prostate cancer is the second leading cause of noncutaneous cancer-related deaths in American men. Androgen deprivation therapy (ADT), radical prostatectomy, and radiotherapy remain the primary treatment for patients with early-stage prostate cancer (castration-sensitive prostate cancer). Following ADT, many patients ultimately develop metastatic castration-resistant prostate cancer (mCRPC). Standard chemotherapy options for CRPC are docetaxel (DTX) and cabazitaxel, which increase median survival, although the development of resistance is common. Cancer stem-like cells possess mesenchymal phenotypes [epithelial-to-mesenchymal transition (EMT)] and play crucial roles in tumor initiation and progression of mCRPC. We have shown that low-dose continuous administration of topotecan (METRO-TOPO) inhibits prostate cancer growth by interfering with key cancer pathway genes. This study utilized bulk and single-cell or whole-transcriptome analysis [(RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq)], and we observed greater expression of several EMT markers, including Vimentin, hyaluronan synthase-3, S100 calcium binding protein A6, TGFB1, CD44, CD55, and CD109 in European American and African American aggressive variant prostate cancer (AVPC) subtypes-mCRPC, neuroendocrine variant (NEPC), and taxane-resistant. The taxane-resistant gene FSCN1 was also expressed highly in single-cell subclonal populations in mCRPC. Furthermore, metronomic-topotecan single agent and combinations with DTX downregulated these EMT markers as well as CD44+ and CD44+/CD133+ "stem-like" cell populations. A microfluidic chip-based cell invasion assay revealed that METRO-TOPO treatment as a single agent or in combination with DTX was potentially effective against invasive prostate cancer spread. Our RNA-seq and scRNA-seq analysis were supported by in silico and in vitro studies, suggesting METRO-TOPO combined with DTX may inhibit oncogenic progression by reducing cancer stemness in AVPC through the inhibition of EMT markers and multiple oncogenic factors/pathways. Significance: The utilization of metronomic-like dosing regimens of topotecan alone and in combination with DTX resulted in the suppression of makers associated with EMT and stem-like cell populations in AVPC models. The identification of molecular signatures and their potential to serve as novel biomarkers for monitoring treatment efficacy and disease progression response to treatment efficacy and disease progression were achieved using bulk RNA-seq and single-cell-omics methodologies.
Subject(s)
Prostatic Neoplasms, Castration-Resistant , Topotecan , Male , Humans , Docetaxel/pharmacology , Topotecan/pharmacology , Prostatic Neoplasms, Castration-Resistant/drug therapy , Administration, Metronomic , Androgen Antagonists/pharmacology , Epithelial-Mesenchymal Transition , Taxoids , Disease Progression , Carrier Proteins/pharmacology , Microfilament Proteins/pharmacologyABSTRACT
PDZ-LIM domain-containing Protein 2 (PDLIM2) has been reported to be downregulated in ovarian cancer. However, its exact function and mechanism in regulating ovarian cancer progression have not been elucidated. This work researched the exert effect and mechanism of PDLIM2 on ovarian cancer progression. Briefly, PDLIM2 expression in clinical tissues of ovarian cancer patients and cells was investigated by qRT-PCR and Western blot. The function of PDLIM2 on the proliferation, colony formation, migration and invasion of ovarian cancer cells was explored via cell counting kit-8, colony formation and Transwell assays. To verify whether PDLIM2 regulates ovarian cancer progression via regulating the transforming growth factor-ß (TGF-ß)/Smad pathway, exogenous TGF-ß (10 ng/mL) treatment was performed on the PDLIM2-overexpressed ovarian cancer cells. PDLIM2 effect on the in vivo growth of ovarian cancer cells was researched by establishing a xenograft tumor model. Immunohistochemistry and Western blot were performed to protein expression in cells and tissues. As a result, PDLIM2 was low-expressed in ovarian cancer tissues/cells. PDLIM2 upregulation attenuated the proliferation, colony formation, migration, invasion and epithelial-mesenchymal transition (EMT) of ovarian cancer cells, and inactivated the TGF-ß/Smad pathway. The opposite results were found in the PDLIM2-silenced ovarian cancer cells. Exogenous TGF-ß treatment abrogated the inhibition of PDLIM2 on the malignant behavior of ovarian cancer cells. PDLIM2 upregulation attenuated the in vivo growth and EMT of ovarian cancer cells. Thus, PDLIM2 attenuates the proliferation, migration, invasion and EMT of ovarian cancer cells via inactivating the TGF-ß/Smad pathway. PDLIM2 may be a usefully target for ovarian cancer treatment.
Subject(s)
Ovarian Neoplasms , Transforming Growth Factor beta , Humans , Female , Transforming Growth Factor beta/metabolism , Cell Movement , Smad Proteins/metabolism , Cell Line, Tumor , Signal Transduction , Epithelial-Mesenchymal Transition , Transforming Growth Factor beta1/metabolism , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Microfilament Proteins/pharmacology , LIM Domain Proteins/genetics , LIM Domain Proteins/metabolism , LIM Domain Proteins/pharmacologyABSTRACT
Bladder cancer, specifically, muscle-invasive bladder cancer (MIBC), is among the most common malignant tumors. Patients with MIBC who cannot tolerate standard drugs require novel treatments. Targeting apoptosis may help treat cancer, which may be achieved with the use of some natural products. Nodosin, found in Isodon serra (Maxim.) Kudo (known as Xihuangcao), may inhibit bladder cancer cells. Transcriptomics and proteomics dual-omic analyses revealed the network pharmacological mechanism: (1) blocking the S phase by up-regulating RPA2, CLSPN, MDC1, PDCD2L, and E2F6 gene expressions, suppressing cancer cell proliferation; (2) inducing apoptosis and autophagy and restraining ferroptosis by up-regulating HMOX1, G0S2, SQSTM1, FTL, SLC7A11, and AIFM2 gene expressions; (3) preventing cancer cell migration by down-regulating NEXN, LIMA1, CFL2, PALLD, and ITGA3 gene expressions. In vivo, nodosin inhibited bladder cancer cell growth in a model of xenograft tumor in nude mice. This study is the first to report basic research findings on the network pharmacological mechanism of cytotoxicity of bladder cancer cells by nodosin, providing novel evidence for the application of nodosin in the field of oncology; however, other mechanisms may be involved in the effects of nodosin for further research. These findings provide a foundation for the development of novel MIBC drugs.
Subject(s)
Biological Products , Urinary Bladder Neoplasms , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/pharmacology , Adaptor Proteins, Signal Transducing/therapeutic use , Animals , Biological Products/pharmacology , Cell Line, Tumor , Cell Proliferation , Cytoskeletal Proteins/metabolism , Cytoskeletal Proteins/pharmacology , Cytoskeletal Proteins/therapeutic use , Diterpenes , Humans , Mice , Mice, Nude , Microfilament Proteins/metabolism , Microfilament Proteins/pharmacology , Microfilament Proteins/therapeutic use , Muscles/metabolism , Muscles/pathology , Network Pharmacology , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathologyABSTRACT
Autism Spectrum Disorders (ASD) are neurodevelopmental disorders whose diagnosis relies on deficient social interaction and communication together with repetitive behavior. To date, no pharmacological treatment has been approved that ameliorates social behavior in patients with ASD. Based on the excitation/inhibition imbalance theory of autism, we hypothesized that bromide ions, long used as an antiepileptic medication, could relieve core symptoms of ASD. We evaluated the effects of chronic sodium bromide (NaBr) administration on autistic-like symptoms in three genetic mouse models of autism: Oprm1-/-, Fmr1-/- and Shank3Δex13-16-/- mice. We showed that chronic NaBr treatment relieved autistic-like behaviors in these three models. In Oprm1-/- mice, these beneficial effects were superior to those of chronic bumetanide administration. At transcriptional level, chronic NaBr in Oprm1 null mice was associated with increased expression of genes coding for chloride ions transporters, GABAA receptor subunits, oxytocin and mGlu4 receptor. Lastly, we uncovered synergistic alleviating effects of chronic NaBr and a positive allosteric modulator (PAM) of mGlu4 receptor on autistic-like behavior in Oprm1-/- mice. We evidenced in heterologous cells that bromide ions behave as PAMs of mGlu4, providing a molecular mechanism for such synergy. Our data reveal the therapeutic potential of bromide ions, alone or in combination with a PAM of mGlu4 receptor, for the treatment of ASDs.
Subject(s)
Autism Spectrum Disorder , Autistic Disorder , Animals , Autism Spectrum Disorder/drug therapy , Autistic Disorder/drug therapy , Behavior, Animal , Bromides/pharmacology , Bromides/therapeutic use , Disease Models, Animal , Fragile X Mental Retardation Protein , Mice , Mice, Knockout , Microfilament Proteins/pharmacology , Microfilament Proteins/therapeutic use , Nerve Tissue Proteins/genetics , Receptors, GABA-A , Social Behavior , Sodium CompoundsABSTRACT
Zearalenone (ZEN) is a worldwide fusarotoxin that poses a threat to the consumer due to its chronic toxicity. Herein we examined the effects of ZEN on adult mouse testis, focusing on oxidative stress, biochemical and morphological parameters. In addition, since cytoskeletal remodeling is a key event for the production of good quality gametes, the expression and localization of two proteins, Dishevelled-associated activator of morphogenesis 1 (DAAM1) and Prolyl endopeptidase (PREP), involved in cytoskeletal dynamics during spermatogenesis were evaluated. To ameliorate the testicular dysfunction induced by ZEN we tested the eventual protective effects of lactic bacteria Lactobacillus plantarum MON03 (LP) on its reprotoxicity. Adult male mice were then treated daily for 2 wks by oral gavage with ZEN and/or LP. The results confirmed that ZEN altered sperm parameters, generated oxidative stress and provoked structural alteration, evidenced by the increased number of abnormal seminiferous tubules and of apoptotic cells, particularly Leydig cells. Interestingly, at molecular level we evaluated, for the first time, the ability of ZEN to alter DAAM1 and PREP protein level and localization. Moreover, the co-treatment with LP, thanks to its capacity to reduce ZEN bioavailability in the gastrointestinal tract, ameliorated all the considered parameters. These results suggest the use of this probiotic as food supplement to prevent/counteract ZEN-induced reprotoxicity.
Subject(s)
Lactobacillales , Zearalenone , Animals , Antioxidants/pharmacology , Male , Mice , Microfilament Proteins/pharmacology , Oxidative Stress , Spermatogenesis , Zearalenone/toxicity , rho GTP-Binding ProteinsABSTRACT
BACKGROUND: mGlu5 metabotropic glutamate receptors are considered as candidate drug targets in the treatment of "monogenic" forms of autism spectrum disorders (ASD), such as Fragile- X syndrome (FXS). However, despite promising preclinical data, clinical trials using mGlu5 receptor antagonists to treat FXS showed no beneficial effects. OBJECTIVE: Here, we studied the expression and function of mGlu5 receptors in the striatum of adult BTBR mice, which model idiopathic forms of ASD, and behavioral phenotype. METHODS: Behavioral tests were associated with biochemistry analysis including qPCR and western blot for mRNA and protein expression. In vivo analysis of polyphosphoinositides hydrolysis was performed to study the mGlu5-mediated intracellular signaling in the striatum of adult BTBR mice under basal conditions and after MTEP exposure. RESULTS: Expression of mGlu5 receptors and mGlu5 receptor-mediated polyphosphoinositides hydrolysis were considerably high in the striatum of BTBR mice, sensitive to MTEP treatment. Changes in the expression of genes encoding for proteins involved in excitatory and inhibitory neurotransmission and synaptic plasticity, including Fmr1, Dlg4, Shank3, Brd4, bdnf-exon IX, Mef2c, and Arc, GriA2, Glun1, Nr2A, and Grm1, Grm2, GriA1, and Gad1 were also found. Behaviorally, BTBR mice showed high repetitive stereotypical behaviors, including self-grooming and deficits in social interactions. Acute or repeated injections with MTEP reversed the stereotyped behavior and the social interaction deficit. Similar effects were observed with the NMDA receptor blockers MK-801 or ketamine. CONCLUSION: These findings support a pivotal role of mGlu5 receptor abnormal expression and function in idiopathic ASD adult forms and unveil novel potential targets for therapy.
Subject(s)
Autism Spectrum Disorder , Mice , Animals , Autism Spectrum Disorder/drug therapy , Autism Spectrum Disorder/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/pharmacology , Nuclear Proteins/therapeutic use , Transcription Factors/metabolism , Mice, Inbred Strains , Corpus Striatum/metabolism , Disease Models, Animal , Microfilament Proteins/metabolism , Microfilament Proteins/pharmacology , Microfilament Proteins/therapeutic use , Nerve Tissue Proteins , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Fragile X Mental Retardation Protein/therapeutic useABSTRACT
Microvilli are finger-like membrane protrusions, supported by the actin cytoskeleton, and found on almost all cell types. A growing body of evidence suggests that the dynamic lymphocyte microvilli, with their highly curved membranes, play an important role in signal transduction leading to immune responses. Nevertheless, challenges in modulating local membrane curvature and monitoring the high dynamicity of microvilli hampered the investigation of the curvature-generation mechanism and its functional consequences in signaling. These technical barriers have been partially overcome by recent advancements in adapted super-resolution microscopy. Here, we review the up-to-date progress in understanding the mechanisms and functional consequences of microvillus formation in T cell signaling. We discuss how the deformation of local membranes could potentially affect the organization of signaling proteins and their biochemical activities. We propose that curved membranes, together with the underlying cytoskeleton, shape microvilli into a unique compartment that sense and process signals leading to lymphocyte activation.
Subject(s)
Cell Membrane/immunology , Lymphocyte Activation/physiology , Microvilli/immunology , Signal Transduction/immunology , T-Lymphocytes/ultrastructure , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/ultrastructure , Animals , Carrier Proteins/pharmacology , Cell Line , Cell Membrane/drug effects , Cell Membrane/ultrastructure , Cell Shape , Cyclodextrins/pharmacology , Cytokines/physiology , Glycocalyx/drug effects , Glycocalyx/immunology , Humans , Lymphocyte Activation/drug effects , Membrane Lipids/immunology , Membrane Proteins/immunology , Mice , Microfilament Proteins/pharmacology , Microscopy, Electron, Scanning , Microvilli/drug effects , Microvilli/ultrastructure , Receptors, Antigen, T-Cell/immunology , Signal Transduction/drug effects , Stress, Mechanical , Surface Properties , Synaptosomes/drug effects , Synaptosomes/immunology , Synaptosomes/ultrastructure , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
Endothelial progenitor cells (EPCs) are bone-marrow derived cells that are critical in the maintenance of endothelial wall integrity and protection of ischemic myocardium through the formation of new blood vessels (vasculogenesis) or proliferation of pre-existing vasculature (angiogenesis). Diabetes mellitus (DM) and the metabolic syndrome are commonly associated with ischemic heart disease through its pathological effects on the endothelium and consequent endothelial dysfunction. Thymosin-ß4 (Tß4) which expressed in the embryonic heart is critical in epicardial and coronary artery formation. In this study, we explored the effects of Tß4 treatment on diabetic EPCs in vitro and intramyocardial injection of Tß4-treated and non-Tß4 treated EPCs following acute myocardial infarction (MI) of diabetic rats in vivo. It was found that 10 ng/mL Tß4 increased migration, tubule formation, and angiogenic factor secretion of diabetic EPCs in vitro. In vivo, although implantation of Tß4 treated diabetic EPCs significantly increased capillary density and attracted more c-Kit positive progenitor cells into the infarcted hearts as compared with implantation of non-Tß4 treated diabetic EPCs, the significantly improved left ventricular ejection fraction was only found in the rats which received non-Tß4 treated EPCs. The data suggests that a low dose Tß4 increases diabetic EPC migration, tubule formation, and angiogenic factor secretion. However, it did not improve the effects of EPCs on left ventricular pump function in diabetic rats with MI.
Subject(s)
Diabetes Mellitus, Experimental/therapy , Echocardiography/methods , Endothelial Progenitor Cells/transplantation , Microfilament Proteins/therapeutic use , Myocardial Infarction/therapy , Thymosin/therapeutic use , Animals , Disease Models, Animal , Humans , Male , Microfilament Proteins/pharmacology , Obesity , Rats , Rats, Zucker , Thymosin/pharmacologyABSTRACT
Intra-abdominal infection is the second most common cause of sepsis, and the mortality rate from abdominal sepsis remains high. High molecular weight (HMW) hyaluronic acid (HA) has been studied in sterile injury models as an anti-inflammatory and anti-permeability agent. This study evaluated the therapeutic effects of intraperitoneal HMW HA administration in mice with peritonitis-induced sepsis. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP), followed 4âh later by an intraperitoneal injection of HMW HA (20âmg/kg) solution or phosphate buffered saline (PBS). Survival, physiological data, organ injury, bacterial burden, and inflammatory cytokine levels were assessed in the CLP mice. To assess the effect of HA on macrophage phagocytosis activity, RAW264.7 cells, primed with lipopolysaccharide, were exposed with either PBS or HMW HA (500âµg/mL) prior to exposure to 10 CFU of E coli bacteria. HMW HA instillation significantly improved blood oxygenation, lung histology, and survival in CLP mice. Inflammatory cytokine levels in the plasma and bacterial burdens in the lung and spleen were significantly decreased by HA administration at 24âh after CLP. At 6âh after CLP, HA significantly decreased bacterial burden in the peritoneal lavage fluid. HMW HA administration significantly increased E coli bacterial phagocytosis by RAW264.7 cells in part through increased phosphorylation of ezrin/radixin/moesin, a known downstream target of CD44 (a HA receptor); ezrin inhibition abolished the enhanced phagocytosis by RAW264.7 cells induced by HA. Intraperitoneal administration of HMW HA had therapeutic effects against CLP-induced sepsis in terms of suppressing inflammation and increasing antimicrobial activity.
Subject(s)
Hyaluronic Acid/therapeutic use , Peritonitis/complications , Peritonitis/drug therapy , Sepsis/drug therapy , Sepsis/etiology , Animals , Cecum/injuries , Cytoskeletal Proteins/pharmacology , Ligation/adverse effects , Male , Membrane Proteins/pharmacology , Mice , Mice, Inbred C57BL , Microfilament Proteins/pharmacology , Phosphorylation/drug effects , Punctures/adverse effects , RAW 264.7 CellsABSTRACT
MDR and tumor migration and invasion are still the main obstacles to effective breast cancer chemotherapies. Transgelin 2 has recently been shown to induce drug resistance, tumor migration, and invasion. The aim of this study was to determine the biological functions of Transgelin 2 and the mechanism underlying how Transgelin 2 induces paclitaxel (PTX) resistance and the migration and invasion of breast cancer. We detected that the protein level of Transgelin 2 was significantly upregulated in breast cancer tissues compared with adjacent nontumor tissues. A bioinformatics analysis indicated that Transgelin 2 was significantly related to clinicopathologic parameters and patient prognosis. Overexpression of Transgelin 2 enhanced the migration and invasion of human breast cancer cells and decreased the sensitivity of breast cancer cells to paclitaxel. Meanwhile, the tumorigenesis and metastasis of breast cancer cells were also enhanced by Transgelin 2 overexpression in vivo Moreover, Transgelin 2 overexpression activated the PI3K/Akt/GSK-3ß pathway by increasing the phosphorylation levels of Akt and GSK-3ß and decreasing the expression of PTEN. We also found that Transgelin 2 could directly interact with PTEN and was located upstream of PTEN. Furthermore, the PI3K/Akt pathway inhibitor MK-2206 reversed the resistance to paclitaxel and inhibited the migration and invasion of breast cancer cells. These findings indicate that Transgelin 2 promotes paclitaxel resistance and the migration and invasion of breast cancer by directly interacting with PTEN and activating the PI3K/Akt/GSK-3ß pathway. Transgelin 2 may therefore be useful as a novel biomarker and therapeutic target for breast cancer.
Subject(s)
Breast Neoplasms/drug therapy , Glycogen Synthase Kinase 3 beta/metabolism , Microfilament Proteins/therapeutic use , Muscle Proteins/therapeutic use , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Apoptosis , Cell Line, Tumor , Cell Movement , Female , Humans , Mice , Mice, Nude , Microfilament Proteins/pharmacology , Muscle Proteins/pharmacologyABSTRACT
During the development of mammalian cortex, late neurons generated by neuronal progenitors bypass earlier-born neurons and migrate to reach upper layers of cortical plate in an inner-to-outer fashion. Filamentous-actin (F-actin) can regulate neuronal migration, whereas Coactosin-like protein 1 (Cotl1) modulates F-actin. Lys 75 and Arg 73 of Cotl1 play an important role in binding F-actin; when they are mutated to Glu, Cotl1 cannot bind F-actin, called as a non-actin-binding mutant (ABM). The Lys 131 site of Cotl1, the 5-Lipoxygenase (5LO) binding site, is spatially close to Lys 75, leading to impact the binding of Cotl1 to F-actin. When Lys 131 is mutated to Ala (K131A), Cotl1 cannot bind to 5LO. We have demonstrated that overexpression of Cotl1 inhibited neuronal migration and increased the length of neuronal leading processes. To further explore cellular and molecular mechanisms of Cotl1's effect on neuronal migration, we constructed two mutant vectors-Cotl1-ABM and Cotl1-K131A and studied using in utero electroporation and primary neuronal culture technique. Results indicated that in the Cotl1-ABM group, the neuronal migration and length of the leading process both recovered as control neurons at the postnatal day 1 (P1), while in the Cotl1-K131A group, numerous neurons remained in deeper layers of cortical plate or intermediate zone. However, at P7, most Cotl1-K131A transfected neurons reached their destination. Moreover, we found that overexpression of Cotl1 inhibited the proliferation and mitotic activity of NPs. Therefore, These results demonstrated that Cotl1 played an important role in mouse neocortical development.
Subject(s)
Cell Movement , Microfilament Proteins/pharmacology , Neocortex/cytology , Neurons/cytology , Animals , Cell Proliferation , Mice , Mitosis , Stem Cells/cytologyABSTRACT
Thymosin beta 4 (Tß4), an actin-sequestering protein, is involved in tissue development and regeneration. It prevents inflammation and fibrosis in several tissues. We investigated the role of Tß4 in chronic ethanol- and acute lipopolysaccharide- (LPS-) induced mouse liver injury. C57BL/6 mice were fed 5% ethanol in liquid diet for 4 weeks plus binge ethanol (5 g/kg, gavage) with or without LPS (2 mg/kg, intraperitoneal) for 6 hours. Tß4 (1 mg/kg, intraperitoneal) was administered for 1 week. We demonstrated that Tß4 prevented ethanol- and LPS-mediated increase in liver injury markers as well as changes in liver pathology. It also prevented ethanol- and LPS-mediated increase in oxidative stress by decreasing ROS and lipid peroxidation and increasing the antioxidants, reduced glutathione and manganese-dependent superoxide dismutase. It also prevented the activation of nuclear factor kappa B by blocking the phosphorylation of the inhibitory protein, IκB, thereby prevented proinflammatory cytokine production. Moreover, Tß4 prevented fibrogenesis by suppressing the epigenetic repressor, methyl-CpG-binding protein 2, that coordinately reversed the expression of peroxisome proliferator-activated receptor-γ and downregulated fibrogenic genes, platelet-derived growth factor-ß receptor, α-smooth muscle actin, collagen 1, and fibronectin, resulting in reduced fibrosis. Our data suggest that Tß4 has antioxidant, anti-inflammatory, and antifibrotic potential during alcoholic liver injury.
Subject(s)
Chemical and Drug Induced Liver Injury/drug therapy , Ethanol/adverse effects , Fibrosis/drug therapy , Inflammation/drug therapy , Lipopolysaccharides/adverse effects , Microfilament Proteins/therapeutic use , Oxidative Stress/drug effects , Thymosin/therapeutic use , Animals , Chemical and Drug Induced Liver Injury/pathology , Female , Mice , Mice, Inbred C57BL , Microfilament Proteins/pharmacology , Thymosin/pharmacologyABSTRACT
BACKGROUND AND OBJECTIVE: Human periodontal ligament cells (HPDLCs) express transforming growth factor-ß1 (TGF-ß1) that regulates differentiation and proliferation, and plays key roles in homeostasis of PDL tissue. Transgelin is a cytoskeleton-associated protein with an Smad-binding element in its gene promoter region. In this study, we examined the localization and potential function of transgelin in PDL tissue and cells. MATERIAL AND METHODS: Microarray analysis of HPDLC lines (2-14, 2-23 and 2-52) was performed. Expression of transgelin in HPDLCs was examined by quantitative reverse transcription-polymerase chain reaction, immunofluorescence staining and western blot analysis. Effects of TGF-ß1 and its signaling inhibitor, SB431542, on transgelin expression in HPDLCs were examined by western blot analysis. The effects of transgelin knockdown by small interfering RNA (siRNA) on HPDLC proliferation stimulated by TGF-ß1 were assessed by WST-1 assay. RESULTS: In microarray and quantitative reverse transcription-polymerase chain reaction analyses, the expression levels of transgelin (TAGLN) in 2-14 and 2-23 cells, which highly expressed PDL markers such as periostin (POSTN), tissue non-specific alkaline phosphatase (ALPL), α-smooth muscle actin (ACTA2) and type I collagen A1 (COL1A1), was significantly higher than those in 2-52 cells that expressed PDL markers weakly. Immunohistochemical and immunofluorescence staining revealed expression of transgelin in rat PDL tissue and HPDLCs. In HPDLCs, TGF-ß1 treatment upregulated transgelin expression, whereas inhibition of the type 1 TGF-ß1 receptor by SB431542 suppressed this upregulation. Furthermore, TAGLN siRNA transfection did not promote the proliferation of HPDLCs treated with TGF-ß1. The expression levels of CCNA2 and CCNE1, which regulate DNA synthesis and mitosis through the cell cycle, were also not upregulated in HPDLCs transfected with TAGLN siRNA. CONCLUSION: Transgelin is expressed in PDL tissue and might have a role in HPDLC proliferation induced by TGF-ß1 stimulation.
Subject(s)
Microfilament Proteins/pharmacology , Muscle Proteins/pharmacology , Periodontal Ligament/drug effects , Transforming Growth Factor beta/antagonists & inhibitors , Adult , Benzamides/pharmacology , Blotting, Western , Cell Proliferation/drug effects , Cells, Cultured , Dioxoles/pharmacology , Female , Fluorescent Antibody Technique , Humans , Male , Microfilament Proteins/physiology , Muscle Proteins/physiology , Periodontal Ligament/cytology , Reverse Transcriptase Polymerase Chain Reaction , Tissue Array Analysis , Transforming Growth Factor beta/pharmacology , Young AdultABSTRACT
Designing bacterial vectors for cancer therapy represents a major challenge. Recent studies have explored novel strategies to balance benefit and safety. A study by Mercado-Lubo et al. has developed a next-generation concept combining bacterial properties with nanoparticles, demonstrating efficacy in combination with chemotherapeutics.
Subject(s)
Biomimetics/methods , Drug Delivery Systems/methods , Nanomedicine/methods , Salmonella typhimurium/cytology , Bacterial Proteins/pharmacology , Biological Therapy/methods , Drug Therapy , Microfilament Proteins/pharmacology , Nanoparticles/chemistry , Nanoparticles/therapeutic use , Neoplasms/drug therapy , Organisms, Genetically Modified , Salmonella typhimurium/geneticsABSTRACT
PURPOSE: To investigate the glial response of the rat retina to single or repeated intravitreal injections (IVI). METHODS: Albino Sprague-Dawley rats received one or three (one every 7 days) IVI of anti-rat VEGF (5 µL; 0.015 µg/µL), triamcinolone (2.5 or 5 µL; 40 µg/µL; Trigón Depot), bevacizumab (5 µL; 25 µg/µL; Avastin), or their vehicles (PBS and balanced salt solution) and were processed 7 days after the last injection. Retinas were dissected as whole mounts and incubated with antibodies against: Iba1 (Ionized Calcium-Binding Adapter Molecule 1) to label retinal microglia, GFAP (Glial Fibrillary Acidic Protein) to label macroglial cells, and vimentin to label Müller cells. The retinas were examined with fluorescence and confocal microscopy, and the numbers of microglial cells in the inner retinal layers were quantified using a semiautomatic method. RESULTS: All the injected substances caused an important micro- and macroglial response locally at the injection site and all throughout the injected retina that was exacerbated by repeated injections. The microglial response was also observed but was milder in the contralateral noninjected eyes. The IVI of the humanized antibody bevacizumab caused a very strong microglial reaction in the ipsilateral retina. Two types of macroglial response were observed: astrocyte hypertrophy and Müller end-foot hypertrophy. While astrocyte hypertrophy was widespread throughout the injected retina, Müller end-foot hypertrophy was localized and more extensive with triamcinolone use or after repeated injections. CONCLUSIONS: Intravitreal injections cause micro- and macroglial responses that vary depending on the injected agent but increase with repeated injections. This inflammatory glial response may influence the effects of the injected substances on the retina.
Subject(s)
Neuroglia/drug effects , Retina/drug effects , Triamcinolone/pharmacology , Vascular Endothelial Growth Factor A/pharmacology , Angiogenesis Inhibitors/pharmacology , Animals , Antibodies/metabolism , Bevacizumab/pharmacology , Calcium-Binding Proteins/pharmacology , Fluorescent Antibody Technique , Glial Fibrillary Acidic Protein/pharmacology , Intravitreal Injections , Microfilament Proteins/pharmacology , Microglia/drug effects , Rats, Sprague-Dawley , Triamcinolone/administration & dosage , Vascular Endothelial Growth Factor A/administration & dosage , Vascular Endothelial Growth Factor A/immunology , Vimentin/pharmacologyABSTRACT
Host actin cytoskeleton polymerization has been shown to play an important role during Trypanosoma cruzi internalization into mammalian cell. The structure and dynamics of the actin cytoskeleton in cells are regulated by a vast number of actin-binding proteins. Here we aimed to verify the impact of AFAP-1L1, during invasion and multiplication of T. cruzi. Knocking-down AFAP-1L1 increased parasite cell invasion and intracellular multiplication. Thus, we have shown that the integrity of the machinery formed by AFAP-1L1 in actin cytoskeleton polymerization is important to hinder parasite infection.
Subject(s)
Actin Cytoskeleton/metabolism , Adaptor Proteins, Signal Transducing/pharmacology , Microfilament Proteins/pharmacology , Trypanosoma cruzi/drug effects , Animals , Chagas Disease/parasitology , Cytoplasm/metabolism , Female , Humans , Male , Mice , Mice, Inbred C57BL , Microfilament Proteins/metabolism , Trypanosoma cruzi/metabolismABSTRACT
The goal of this work was to elucidate the mechanism of inhibition of the actin-activated ATPase of myosin subfragment-1 (S1) by the calponin-like protein from mussel bivalve muscle. The calponin-like protein (Cap) is a 40-kDa actin-binding protein from the bivalve muscle of the mussel Crenomytilus grayanus. Kinetic parameters Vmax and KATPase of actomyosin ATPase in the absence and the presence of Cap were determined to investigate the mechanism of inhibition. It was found that Cap mainly causes increase in KATPase value and to a lesser extent the decrease in Vmax, which indicates that it is most likely a competitive inhibitor of actomyosin ATPase. Analysis of Vmax and KATPase parameters in the presence of tropomyosin revealed that the latter is a noncompetitive inhibitor of the actomyosin ATPase.
Subject(s)
Calcium-Binding Proteins/pharmacology , Microfilament Proteins/pharmacology , Muscle Proteins/pharmacology , Myosins/antagonists & inhibitors , Mytilidae , Animals , Kinetics , Muscle, Smooth , CalponinsABSTRACT
BACKGROUND: Random skin flaps can be used throughout the hands and fingers. Thymosin ß4 can increase blood flow and reduce ischemia-reperfusion injury; the study was undertaken to investigate the effect of thymosin ß4 on the survival of random skin flaps. METHODS: A total of 45 male Sprague-Dawley rats were used and subjected to a random-pattern skin flaps operation. Rats were randomly divided into three groups: a control group (group A: intraperitoneal injection of saline, 5 mg/kg/d) and two treatment groups (group B: intraperitoneal injection of thymosin ß4, a single 5 mg/kg dose per day) and (group C: intraperitoneal injection of thymosin ß4, 5 mg/kg dose twice per day). The flap surviving area was measured after 7 days, and tissue samples were stained with hematoxylin and eosin. Vascular endothelial growth factor (VEGF) expression was determined using immunohistochemical methods. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were examined with kits. RESULTS: Thymosin ß4 significantly reduced the necrotic area in the treatment groups after 7 days compared with the control group, and the rats receiving thymosin ß4 5 mg/kg twice per day had the highest survival rate. VEGF expression and SOD activity markedly increased in the treatment groups compared with the control group, whereas MDA levels were lower in the treatment groups than in the control group. CONCLUSION: Thymosin ß4 may have a dose-dependent effect to promote the survival of random skin flaps.
Subject(s)
Microfilament Proteins/pharmacology , Reperfusion Injury/prevention & control , Thymosin/pharmacology , Thymosin/therapeutic use , Animals , Dose-Response Relationship, Drug , Immunohistochemistry , Injections, Intraperitoneal , Male , Microvessels/metabolism , Rats, Sprague-Dawley , Regional Blood Flow/physiology , Superoxide Dismutase/metabolism , Surgical Flaps , Vascular Endothelial Growth Factor A/metabolismABSTRACT
In somatic cells, the position of the cell centroid is dictated by the centrosome. The centrosome is instrumental in nucleus positioning, the two structures being physically connected. Mouse oocytes have no centrosomes, yet harbour centrally located nuclei. We demonstrate how oocytes define their geometric centre in the absence of centrosomes. Using live imaging of oocytes, knockout for the formin 2 actin nucleator, with off-centred nuclei, together with optical trapping and modelling, we discover an unprecedented mode of nucleus positioning. We document how active diffusion of actin-coated vesicles, driven by myosin Vb, generates a pressure gradient and a propulsion force sufficient to move the oocyte nucleus. It promotes fluidization of the cytoplasm, contributing to nucleus directional movement towards the centre. Our results highlight the potential of active diffusion, a prominent source of intracellular transport, able to move large organelles such as nuclei, providing in vivo evidence of its biological function.